The invention features biopolymer foams, composite biopolymer foams, biocompatible constructs comprising biopolymer foams and extracellular matrix particulates and methods for making and using these foams and foam compositions. The foams and foam compositions can be used in vitro, for example, for model systems for research, or in vivo as prostheses or implants to replace damaged or diseased tissues or to provide scaffolds which, when occupied by cells, e.g., host cells, are remodeled to become functional tissues. In either case, the foams and foam compositions can be seeded with cells, e.g., mammalian cells, e.g., human cells, of the same type as those of the tissue which the foams or foam compositions is used to repair or reconstruct. Examples of tissues which can be repaired and/or reconstructed using the foams and foam compositions described herein include nervous tissue, skin, vascular tissue, muscle tissue, connective tissue such as bone, cartilage, tendon, and ligament, kidney tissue, and glandular tissue such as liver tissue and pancreatic tissue. In one embodiment, the foams and foam compositions seeded with tissue specific cells are introduced into a recipient, e.g., a mammal, e.g., a human. Alternatively, the seeded cells which have had an opportunity to organize into a tissue in vitro and to secrete tissue specific biosynthetic products such as extracellular matrix proteins and/or growth factors which bond to the foams and foam compositions are removed prior to introduction of the foams and foam compositions into a recipient.
Accordingly, the invention pertains to single density biopolymer foams having selected characteristics. In a preferred embodiment, the single density biopolymer foams comprise a network of communicating microcompartments having biopolymer molecules and/or biopolymer filaments interspersed within the walls of the microcompartments. The microcompartments of these foams typically have volume dimensions of x, y, and z, wherein x=length, y=width, and z=height, are substantially equal, and range from about 1 .mu.m to about 300 .mu.m, preferably from about 50 .mu.m to about 100 .mu.m. The average wall thicknesses of the microcompartments of the single density biopolymer foams is less than about 10 .mu.m. Examples of biopolymers which can be used in the single density biopolymer foams include collagen, alginic acid, polyvinyl alcohol, elastin, chondroitin sulfate, laminin, fibronectin, fibrinogen, and combinations of these biopolymers. A preferred biopolymer is collagen, e.g., porcine fetal collagen. In other embodiments, the single density biopolymer foams can include extracellular matrix particulates and/or cells.
Single density biopolymer foams of the invention can be prepared by forming a biopolymer solution, crosslinking the biopolymer in the biopolymer solution, and freeze-drying the biopolymer solution to form a single density biopolymer foam. In another embodiment, the crosslinking step occurs after the freeze-drying step. In a preferred embodiment, the method also includes a step, prior to the crosslinking step, of polymerizing the biopolymer in the biopolymer solution to form a biopolymer lattice. When collagen, the preferred biopolymer, is used in this method, it can be crosslinked by priming with lysyl oxidase. To reduce splitting of the foam, the biopolymer can also be freeze-dried in the presence of an anti-freeze polypeptide, e.g. a type I, I, or III anti-freeze polypeptide, or an anti-freeze glycoprotein. The invention also pertains to single density biopolymer foams prepared by this method.
The invention also pertains to double density biopolymer foams having selected characteristics. In a preferred embodiment, the double density biopolymer foams comprise a network of communicating microcompartments having biopolymer molecules and/or biopolymer filaments interspersed within the walls of the microcompartments. The microcompartments of these foams typically have volume dimensions of x, y, and z, wherein x=length, y=width, and z=height and two of which are substantially equal and range from about 1 .mu.m to about 300 .mu.m and the third of which is less than the two dimensions which are substantially equal. The average wall thicknesses of the microcompartments of the double density biopolymer foams is less than about 10 .mu.m. Preferred biopolymers for use in double density foams are described herein. In other embodiments, the double density biopolymer foams can include extracellular matrix particulates and/or cells.
Double density biopolymer foams of the invention can be prepared by forming a biopolymer solution and then crosslinking the biopolymer in the biopolymer solution. The biopolymer solution can then be freeze-dried to form a foam, hydrated, and shaped to have a selected form. The foam having the selected form can then be dried to yield the double density biopolymer foam. In another embodiment, the crosslinking step occurs after the freeze-drying step. In a preferred embodiment, the method for preparing double density biopolymer foams includes, prior to the crosslinking step, the step of polymerizing the biopolymer in the biopolymer solution to form a biopolymer lattice. The invention also pertains to double density biopolymer foams prepared by this method.
Composite biopolymer foams which include both single and double density foams are also specifically contemplated by the invention. The foams or composite foams can further be conditioned with cells prior to use in vitro or in vivo. Composite biopolymer foams are formed by first providing a double density biopolymer foam and then hydrating the double density biopolymer foam with, for example, water or a biopolymer solution. A biopolymer solution is then added to the hydrated double density biopolymer foam and the solution and hydrated double density foam are freeze-dried to form a composite biopolymer foam. Prior to the freeze-drying step, the biopolymer in the biopolymer foam can be crosslinked. The invention also includes composite biopolymer foams prepared by this method. The single density and double density foams of the composite biopolymer foam can also be freeze-dried after cell conditioning.
In another aspect, the invention pertains to biocompatible constructs which include single or double density biopolymer foams and extracellular matrix particulates. The extracellular matrix particulates can be dispersed throughout the foam, e.g., the extracellular matrix particulates are included within in a biopolymer solution or suspension which is dispersed throughout the foam and/or which is coated on the surface of the biopolymer foam. The biopolymer foam with the extracellular matrix particulates can then be freeze-dried.
In yet another aspect, the invention pertains to methods for preparing biopolymer-coated, e.g., collagen-coated, single or double density foams. These methods include preparing the single or double density foams by the methods described herein and then applying a biopolymer solution, which can further include extracellular matrix particulates, to the foams, thereby forming a biopolymer-coated foam. After the foam has been coated, it can be freeze-dried.
The invention also pertains to methods for preparing extracellular matrix particulate-coated single or double density foams. These methods include preparing the single or double density foams by the methods described herein and then applying extracellular matrix particulates, e.g., extracellular matrix particulates suspended in a collagen solution, to the foams, thereby forming an extracellular matrix particulate-coated foam. In one embodiment, the coated foam can then be freeze-dried.
The biopolymer foams and foam compositions, with or without extracellular matrix particulates, of the invention can be used, for example, as skin substitutes or skin dressings, vascular implants, orthopedic implants, dental implants, connective tissue implants, e.g., cartilage implants, urological implants, and glandular implants. Typically, the biopolymer foams and foam compositions are conditioned with cells. In a preferred embodiment, the biopolymer foams and foam compositions can be used as skin dressings. The skin dressing can be a composite biopolymer foam which includes a single density collagen foam and a double density collagen foam. The single density biopolymer foam can be conditioned with human dermal fibroblasts and the double density foam can be conditioned with human keratinocytes such that a stratum corneum is formed. After cell conditioning, the single and double density biopolymer foams of the composite can be freeze-dried. In another embodiment, the skin dressing can be a double density biopolymer foam which has dermal fibroblasts dispersed throughout the foam and epidermal cells on one surface of the foam.
The foams and foam compositions of the invention can also be used as vascular prostheses. In one embodiment, the vascular prosthesis is a double density biopolymer foam or a composite biopolymer foam in the form of a tube. In a preferred embodiment, the tubular vascular prosthesis includes endothelial cells on its luminal surface and smooth muscle cells throughout and on its abluminal surface. The vascular prosthesis can also include a layer of adventitial cells on the smooth muscle cells. After cell conditioning, the double density biopolymer foam or composite biopolymer foam of the vascular prosthesis can be freeze-dried.
Orthopedic and dental implants can also be produced from the foam and foam compositions, with or without extracellular matrix particulates, of the invention. Typically, the foam and foam compositions which are used as orthopedic and dental implants include calcium phosphate cement. An example of such a dental implant is an alveolar ridge builder which is composed of a double density biopolymer foam in the form of a tube containing resorbable calcium phosphate cement. Alternatively, the biopolymer foams and foam compositions can be produced to include hydroxyapatite and used, for example, as dental implants. An alveolar ridge substitute which includes a double density biopolymer foam in the form of a tube containing nonresorbable hydroxyapatite is an example of such a dental implant.
Also contemplated herein are dental implants capable of promoting periodontal ligament repair and bone rebuilding and methods for promoting periodontal ligament repair and bone rebuilding using these implants. Typically, these dental implants include an apron shaped double or quadruple density biopolymer foam. In one embodiment, the apron shaped double or quadruple density biopolymer foam includes an outpocketing containing calcium phosphate cement. To promote periodontal ligament repair and bone rebuilding, an area of tooth requiring periodontal ligament repair and bone rebuilding is contacted with the apron shaped foam, e.g., by the tying the strings of the double or quadruple density biopolymer foam around a tooth to secure the apron to an area of tooth requiring periodontal ligament repair and bone rebuilding.
In yet another aspect, the biopolymer foams and foam compositions of the invention can be used as connective tissue implants, e.g., cartilage, tendon, ligament implants. In one embodiment, the foams and foam compositions are prepared as cartilage implants. In a preferred embodiment, the cartilage implants include a substrate including a biopolymer solution and a calcium phosphate cement which has set into a cement and a single or double density biopolymer foam embedded, e.g., by freeze-drying, in one face of the cementous substrate. The single or double density biopolymer foam of the cartilage implant can also be seeded with chondrocytes. In another embodiment, the foams and foam compositions are prepared as ligament implants. Typically, the ligament implants are composed of a plurality of biopolymer filaments and a single or double density biopolymer foam.
In a still further aspect of the invention, the foams and foam compositions are prepared as glandular implants. The glandular implants can be prepared from a foam or foam composition described herein and can include extracellular matrix particulates derived from glandular tissue. In a preferred embodiment, the glandular tissue can also be seeded with the appropriate glandular cells, e.g., pancreatic islet cells, hepatocytes.